A Novel SXT/R391 Integrative and Conjugative Element Carries Two Copies of the blaNDM-1 Gene in Proteus mirabilis

ABSTRACT The rapid spread of the blaNDM-1 gene is a major public health concern. Here, we describe the multidrug-resistant Proteus mirabilis strain XH1653, which contains a novel SXT/R391 integrative and conjugative element (ICE), harboring two tandem copies of blaNDM-1 and 21 other resistance genes. XH1653 was resistant to all antibiotics tested, apart from aztreonam. Whole-genome data revealed that two copies of blaNDM-1 embedded in the ISCR1 element are located in HS4 of the novel ICE, which we named ICEPmiChnXH1653. A circular intermediate of ICEPmiChnXH1653 was detected by PCR, and conjugation experiments showed that the ICE can be transferred to the Escherichia coli strain EC600 with frequencies of 1.5 × 10−7. In the recipient strain, the ICE exhibited a higher excision frequency and extrachromosomal copy number than the ICE in the donor strain. We also observed that the presence of ICEPmiChnXH1653 has a negative impact on bacterial fitness and leads to changes in the transcriptome of the host. In vitro evolution experiments under nonselective conditions showed that the two tandem copies of the ISCR1 element and the ISVsa3 element can be lost during repeated laboratory passage. This is the first report of a novel SXT/R391 ICE carrying two tandem copies of blaNDM-1, which also illustrates the role that ICEs may play as platforms for the accumulation and transmission of antibiotic resistance genes. IMPORTANCE The occurrence of carbapenemase-producing Proteus mirabilis, especially those strains producing NDM-1 and its variants, is a major public health concern worldwide. The integrative conjugative element (ICE) plays an important role in horizontal acquisition of resistance genes. In this study, we characterized a novel SXT/R391 ICE from a clinical P. mirabilis isolate that we named ICEPmiChnXH1653, which contains two tandem copies of the carbapenemase gene blaNDM-1. We performed an integrative approach to gain insights into different aspects of ICEPmiChnXH1653 evolution and biology and observed that ICEPmiChnXH1653 obtained the carbapenemase gene blaNDM-1 by ISCR1-mediated homologous recombination. Our study reveals that the transmission of blaNDM-1 by ISCR1 elements or ICEs may be an important contributor to the carbapenem resistance development across species, which could improve our understanding of horizontal gene transfer in clinical environments.

of carbapenemase-producing P. mirabilis is of particular concern; in particular, strains producing the New Delhi metallo-b-lactamase 1 (NDM-1) make treatment extremely difficult (2,3). NDM-1, an Ambler class B b-lactamase, is the main type of carbapenemase, conferring resistance to almost all b-lactams except monobactams (4). Global dissemination of NDM-1 is a major public health problem (5,6). bla NDM-1 is a chimeric gene that has arisen by a gene fusion event linking the aminoglycoside resistance gene aphA6 to a preexisting metallo-b-lactamase (MBL) gene and is frequently located downstream of a complete or truncated copy of ISAba125 (7). The bla NDM-1 gene has been found in various genetic contexts including prophages (8) but is most often associated with other mobile genetic elements that are responsible for its evolution and rapid transmission (9). For instance, ISCRs (insertion sequences with a common region) are often found in the vicinity of bla NDM-1 and considered one of the routes of the fusion of bla NDM-1 and also involved in the mobilization of bla NDM-1 (7,(9)(10)(11). In addition, horizontal dissemination of bla NDM-1 among diverse species can occur via mobile DNA vectors, mostly conjugative plasmids and integrative conjugative elements (ICEs) (12,13).
ICEs are self-transmissible mobile genetic elements which encode modules that facilitate integration/excision, conjugative transfer, and maintenance (14). The element can therefore be excised from the host chromosome to form a circular intermediate that can be transferred to a recipient cell via conjugation (15). The SXT/R391 family, one of the largest families of ICEs, has a highly conserved core of sequences that mediate the sitespecific integration into the 59 end of the prfC gene (14,16). Except for a conserved backbone, SXT/R391 ICEs frequently contain hot spots (HS1 to HS5) and variable regions (VRI to VRV) that carry genes for antimicrobial resistance and metal tolerance (17,18). Many SXT/R391 ICEs have been found in P. mirabilis, contributing to the dissemination of antimicrobial resistance genes (ARGs) including the cephalosporinase gene bla CMY-2 (19)(20)(21). Most recently, an SXT/R391 ICE carrying the carbapenemase gene bla NDM-1 embedded within a truncated Tn125 was identified in Proteus vulgaris (13).
In this study, we characterized a novel SXT/R391 ICE from a P. mirabilis isolate, named ICEPmiChnXH1653, which contains two tandem copies of bla NDM-1 and 21 other ARGs. An integrative approach, combining bacterial conjugation tests, fitness assays, experimental evolution, genomics, and transcriptomics, allowed us to gain insights into diverse aspects of evolution and biology of ICEPmiChnXH1653 (22).
Genetic structure of ICEPmiChnXH1653. ICEPmiChnXH1653 has a length of 165,609 bp (bases 2138935 to 2304493 in XH1653) and a GC content of 50.0%. It was found integrated into the 59 end of the prfC gene, a common insertion site for ICEs in the SXT/R391 family (Fig. 1B). ICEPmiChnXH1653 consisted of a highly conserved backbone involved in essential functions of SXT/R391 ICEs, such as integration (int and xis), mating pair formation (traLEKBVA), exclusion determination (traG and ees), DNA recombination (bet and exo), and regulation (setR) (24). BLAST analysis showed that ICEPmiChnXH1653 had 100% nucleotide identity at 98% coverage to ICEPmiCHN905 that was found in the P. mirabilis strain MD20140905 isolated from stool samples from diarrhea patients in Beijing, China, in 2014, with two region divergences in VRIII and HS4 ( Fig. 1C) (25), indicating that ICEPmiChnXH1653 might have a common origin with ICEPmiCHN905.
Characterization of VRIII and HS4 region of ICEPmiChnXH1653. ICEPmiChnXH1653 harbored two multidrug resistance (MDR) regions in VRIII and HS4, respectively. VRIII contained six ARGs, including erm(42), floR, strB (three copies), aphA (two copies), strA, and sul2. Structural comparison showed that the formation of this MDR cluster in VRIII was likely due to the abundance of transposases and ISCR2 elements.
The MDR region HS4 is 73.75 kb and harbors 19 ARGs coding for b-lactam, fluoroquinolone, fosfomycin, tetracycline, aminoglycoside, sulfamethoxazole, trimethoprim, streptomycin, phenicol, rifamycin, macrolide, and bleomycin resistance, which are clustered together in an ISPpu12-mediated composite transposon flanked by the 8-bp target site duplication (TSD) TAAAGAAA. According to a BLAST analysis, HS4 in ICEPmiChnXH1653 had 99.92% nucleotide identity at 82% coverage to the HS4 in ICEPmiBCP11 that was found in the P. mirabilis strain BCP11 isolated from a fecal swab of a diseased pig with diarrhea in Sichuan Province of China in November 2016, and with the exception of cfr, it carried all the ARGs that were found in ICEPmiBCP11. The HS4 region of ICEPmiChnXH1653 also has a similar genetic environment as HS4 of ICEPmiBCP11, which suggests that these two ICEs isolated from China share an ISPpu12-mediated region (Fig. 1D). However, there are major differences between them: two copies of the carbapenemase gene bla NDM-1 and the chloramphenicol ARG floR were detected in the HS4 region of ICEPmiChnXH1653. Further analysis of the insertion sequences surrounding bla NDM-1 revealed two tandem copies of an ISCR1element (ISCR1-traF-ble MBL -bla NDM-1 -DISAba125-catB3-arr-3-qacED1-sul1). The sequence of the ISCR1 element in the HS4 region of ICEPmiChnXH1653 showed 99.95% nucleotide identity to plasmid pNDM-PM58 from P. mirabilis (GenBank accession no. KP662515.1). Moreover, the two tandem copies of the ISCR1 element were also seen in Escherichia coli Y5 (99.98% nucleotide identity; GenBank accession no. CP013483) that was reported by our lab in 2016 (26). The floR gene was flanked by ISVsa3 elements with a 222-bp 39-conserved segment (39-CS), indicating that ISVsa3 promotes the dissemination of floR. Two copies of IS26 adjacent to bleO are in the same orientation, while another IS26 and two genes (encoded recombinase family protein and transposase, respectively) lie in the opposite orientation, flanked by identical 8-bp TSDs (GTTCATAC; CGCCGGTG). This indicates that IS26 is involved in the accumulation of resistance genes and the rearrangement of multidrug resistance regions.
Transfer ability of ICEPmiChnXH1653. To test this ability to transfer ICEPmiChnXH1653, conjugation experiments were performed, with the P. mirabilis strain XH1653 as donor and the rifampin-resistant strain E. coli EC600 as recipient. ICEPmiChnXH1653 was successfully transferred to E. coli EC600 with a frequency of 1.5 Â 10 27 transconjugants per recipient cell and chromosomally integrated into the 59 end of prfC. The positive transconjugant, subsequently referred to as XH1814, was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and PCR detection of int, attL, attR, and the carbapenemase gene bla NDM-1 . Antimicrobial susceptibility testing showed XH1814 acquired resistance to all antimicrobials tested, apart from ciprofloxacin ( Table 1). Excision of the ICEPmiChnXH1653 and the presence of a circular form were analyzed using PCR with primers LE4 and RE4 (24). The analysis confirmed occurrence of the circular ICE in XH1653 and XH1814 (Fig. 2), indicating that ICEPmiChnXH1653 could form circular intermediates.  To investigate the biology of ICEPmiChnXH1653, we evaluated three dynamic factors in both the ancestor strain and the recipient strain. Real-time quantitative PCR assays were developed to determine the percentages of int/prfC (mean copy number per cell), attB/attP (extrachromosomal copy number of the ICE), and attB/prfC (the frequency of excision) in a culture (Fig. 3A). The ratio of int/prfC was found to be 0.96 6 0.20 for XH1814 and 0.98 6 0.11 for XH1653, as expected for a single copy of an ICE integrated in the chromosome prfC target. The results of attB/attP showed that ICEPmiChnXH1653 in XH1814 exhibited a relatively higher excision frequency than the ICE in XH1653; this might indicate that the ICE is not as stable in E. coli as it is in the original host. We also found that both XH1814 and XH1653 had multiple copies of extrachromosomal ICEs (21 6 10 and 11 6 3, respectively), suggesting that the circular intermediate of ICEPmiChnXH1653 is capable of replicating in a small subset of the cell population.
Presence of ICEPmiChnXH1653 influences host fitness and transcriptome. To estimate the fitness cost of ICE carriage, we compared growth rates of the transconjugant XH1814 and the ICE-free recipient strain EC600 in Mueller-Hinton (MH) liquid medium. XH1814 containing ICEPmiChnXH1653 exhibited a significantly decreased growth rate compared to the strain without the ICE, EC600 (Fig. 3B), indicating that ICEPmiChnXH1653 confers a fitness cost on the host.
To better understand the molecular basis for the decrease in fitness due to ICEPmiChnXH1653 in EC600, we performed transcriptomic analyses of the strain with and without the ICE (XH1814 versus EC600) using RNA sequencing (RNA-Seq). In comparison to strain EC600, a total of 22 genes were differentially expressed in XH1814 (false-discovery rate [FDR] , 0.05). Among them, nine genes were upregulated, and 13 genes were downregulated (see Table S1 in the supplemental material). The upregulated genes were involved in lipid metabolism and amino acid metabolism, and the downregulated genes were involved in the metabolic pathway, replication and repair pathway, and quorum sensing pathway. Increased transcription of genes encoding 4-oxalomesaconate tautomerase (DK885_16125), bifunctional aldehyde dehydrogenase (DK885_12690), hydrogenase-4 component J (DK885_06720), and galactose-proton symporter (DK885_04375) was noticeable, whereas genes for multidrug resistance protein (MdtL), guanine/hypoxanthine permease (GhxQ), and cold shock protein (CspB) were expressed at lower levels in XH1814. We also compared gene expression levels of EC600 and XH1814 using the average reads per kilobase per million mapped reads (RPKM), with no significant difference between the detectable levels. However, the average RPKM of ICE in XH1814 was higher than the average RPKM of EC600 or XH1814 (P , 0.05) (Fig. 3C).
XH1814 lost carbapenem resistance following repeated laboratory passage. Given the apparent cost of the ICEPmiChnXH1653 carriage, we performed experimental evolution experiments with the transconjugant XH1814 and the ICE-free recipient strain EC600 as a control to identify the putative emergence of compensatory mechanisms associated with ICE carriage. We observed that the strain on day 10, "XH1814D10," had a significantly faster growth than XH1814 and the strain on day 5 (Fig. 4A). However, the ICE-free control strain also showed evolutionary adaptations with significantly faster growth than the strain on day 0 and day 5 (Fig. 4B). As compensatory mutations may not be associated with ICE carriage, we chose not to trace compensatory mutations on the whole-genome level. When testing the presence of ICEPmiChnXH1653 in "XH1814D10" using PCR with bla NDM-1 primers, we observed that the bla NDM-1 gene was lost while still retaining the ICE (Fig. 5A and B). This strain, named XH1815, had become susceptible to imipenem and meropenem (Table 1). Also, the loss of carbapenem resistance was observed, albeit at a low frequency (8.14% of the colonies) (Fig. 4C).
ISCR1 and ISVsa3 elements were deleted in XH1815. The whole genome of XH1815 was sequenced at high accuracy in order to analyze the molecular events that resulted in the deletion of bla NDM-1 . Strain XH1815 was found to harbor the ICE with a size of 148,447 bp, subsequently referred to as ICEPmiChnXH1815, which chromosomally integrated into the 59 end of the prfC gene. Comparative analysis showed ICEPmiChnXH1815 to exhibit sequence coverage of 99% and identity of 100% to ICEPmiChnXH1653. A deletion of two fragments was found in the HS4 region of ICEPmiChnXH1815 (Fig. 6A) and corresponds to the tandem copies of the ISCR1 and ISVsa3 elements that included the carbapenemase gene bla NDM-1 and the chloramphenicol ARG floR, respectively (Fig. 6B). Our results indicate that ICEPmiChnXH1653 obtained the carbapenemase gene by ISCR1-mediated homologous recombination. As observed in ICEPmiChnXH1815, only one copy of the ISVsa3 was found, and the ISVsa3 element (hp-floR-cynR-ISVsa3) was deleted compared to ICEPmiChnXH1653. We also detected the circular intermediate of the ISVsa3 element, which suggests that ISVsa3-mediated transfer of folR had occurred and the circular intermediate hp-floR-cynR-ISVsa3 had inserted at the location of ISVsa3.

DISCUSSION
The observed increase of P. mirabilis strains that are resistant to carbapenem mediated by the bla NDM-1 gene is of concern as only a limited number of antimicrobials remain available for clinical therapy (12). In this study, we isolated an XDR P. mirabilis strain, XH1653, from a patient suffering from a urinary tract infection, which was resistant to all tested antibiotics with the exception of aztreonam. qPCR and sequencing analysis showed that XH1653 carried two copies of bla NDM-1 , which are located in a sofar-undescribed SXT/R391 ICE.
Mobile elements are associated with the formation of clusters containing ARGs in which different determinants that lead to the MDR phenotype are found in close genetic proximity (27). Antimicrobial resistance determinants in ICEPmiChnXH1653 are carried within VRIII and HS4 regions that are integrated within the conserved ICE backbone, and most of them are located within ARG arrays, composed of ARGs or clusters thereof, and mobile genetic elements such as IS elements, transposons, or integrons. The genes floR, strB, strA, and sul2 are frequently found in the VRIII region of SXT/R391 ICE members (28). Also, erm (42) and aphA are observed in the VRIII region of ICEAplChn1 in Actinobacillus pleuropneumoniae (29). Our finding is also the first report of two copies of the resistance gene fragment strB-aphA in VRIII.
The bla NDM-1 gene is mainly and widely spread by an ISAba125-bounded composite transposon Tn125 (9), which is often located on plasmids in Enterobacteriaceae (12,30,31). Recently, Kong et al. reported that bla NDM-1 was embedded in a truncated ISAba125 composite transposon flanked by IS26 in the ICE from P. vulgaris (13). However, the genetic environment around bla NDM-1 in ICEPmiChnXH1653 is different from the ICE in the study by Kong et al., which revealed two tandem copies of an ISCR1 element. A 151-bp truncated ISAba125 is found upstream of bla NDM-1 , while the ISCR1 lies downstream of both copies of the bla NDM-1 gene and is followed by the bleomycin ARG ble MBL and the trpF gene, which are often identified near bla NDM-1 (9). The ISCR1-like elements might be responsible for the mobilization of bla NDM-1 via the rolling-circle replication in Enterobacteriaceae (30,32,33). In our study, we also observed that the two tandem copies of the ISCR1 element (ISCR1-traF-ble MBL -bla NDM-1 -DISAba125-catB3-arr-3-qacED1-sul1) were lost following extended passage under nonselective conditions, which is a clear indication that two tandem copies of the ISCR1 element move into the 39-CS (qacED1/sul1) of a class 1 integron [aac(69)-Ib-cr-bla OXA-1 -catB3-arr3-qacED1-sul1] by homologous recombination, resulting in the transmission of bla NDM-1 . Homologous recombination is likely to contribute extensively to the duplication of ARGs when no selective pressure is applied. It is possible that the a single ISCR1 element mobilizes the bla NDM-1 gene to move into the 39-CS by rolling-circle transposition and that after attachment, subsequent homologous recombination may result in a duplication of the ARG (34). A similar structure of the two tandem copies of the ISCR1 element was also observed in the chromosome of E. coli Y5 (GenBank accession no. CP013483) (26), and one copy of the ISCR1 element appears to be intact in the plasmid from P. mirabilis (GenBank accession no. KP662515), suggesting a potential translocation of the bla NDM-1 gene between different mobile genetic elements (ICEs and plasmids) and the integration into the chromosome. To the best of our knowledge, this is the first description of two copies of bla NDM-1 embedded within the ISCR1 element, not only in the SXT/R391 element but also in P. mirabilis. ISVsa3 belongs to the ISCR2 family; two copies of the ISVsa3 in the same orientation were found in the HS4 region of ICEPmiChnXH1653, which contained the chloramphenicol ARG floR. He et al. reported that ISVsa3 is able to mediate the transposition of tet(X)-carrying cassettes and that the circular intermediate was able to insert at the location of ISVsa3 on the plasmid (35). We found that the ISVsa3 element (hp-floR-cynR-ISVsa3) was lost during the serial passage in the laboratory, indicating the ISVsa3-mediated transfer of floR had occurred and ISCR elements played a major role for the host bacteria in the mobilization and accumulation of antibiotic resistance genes. After the deletion of the ISCR1 and ISVsa3 element in ICEPmiChnXH1653, the HS4 of the ICE exhibited a high similarity to the HS4 of ICEPmiBCP11 with a coverage of 95% (increase from 82% to 95%) and a sequence identity of 99.95%, contributing to our knowledge of how the dissemination of an ISPpu12-mediated composite MDR transposon in different P. mirabilis strains occurs. A general observation is that the ICEPmiChnXH1653 has been highly efficient in recruiting antimicrobial resistance traits. It has been suggested that recent MDR members of the SXT/R391 family could have evolved from a common ancestor through stepwise integration of horizontally acquired ARG arrays into the conserved backbone.
Here, we performed an integrative approach to gain insights into different aspects of ICEPmiChnXH1653 evolution and biology. An intergenus transfer of ICEPmiChnXH1653 from P. mirabilis to E. coli EC600 at a frequency of 1.5 Â 10 27 was observed, which is relatively low for SXT/R391 (22). The ICEPmiChnXH1653 in the recipient strain exhibited a higher excision frequency and extrachromosomal copy number than the ICE in the ancestor strain. As expected for one attP site on the circular ICE resulting in one unoccupied attB site on the chromosome (attP/attB = 1), an increase in the copy number of attP/attB would indicate that the excised ICE replicates more frequently than the chromosome (36). The extrachromosomal autonomous replication appears to be common for ICEs, as our results for attP/attB were consistent with previous reports (36,37). The relatively higher excision frequency in recipient strain EC600 may be caused by a genetic instability after entering a new host cell. We also found that the acquisition of ICEPmiChnXH1653 results in a fitness cost for the ICE-free recipient strain EC600. Here, several genes showed altered transcription in EC600 after the acquisition of ICE. This number was lower than a report which described that a total of 161 genes were differentially expressed in Pseudomonas putida with the ICEclc (38). The presence of the ICEclc can influence a number of cellular pathways, resulting in direct benefits but also in indirect costs for P. putida (38). The difference of the impact on the transcription of the bacteria might be caused by the genetic interrelationship of the strains used in the studies: the original host of ICEclc is Pseudomonas knackmussii B13, with the new host in the study by Miyazaki et al. (38) belonging to the same genus. However, in our project we used EC600 as the new host for the P. mirabilis XH1653-derived element ICEPmiChnXH1653. Interestingly, the genes carried in ICEPmiChnXH1653 showed a higher expression in XH1814 compared to the average expression in EC600 or XH1814, confirming ICEPmiChnXH1653 activation in XH1814, possibly explaining the burden in fitness.
In conclusion, this is the first report of a novel SXT/R391 ICE carrying two tandem copies of bla NDM-1 . The genetic environment of bla NDM-1 was identical to that of the previously reported bla NDM-1 -carrying plasmid of P. mirabilis PM58 and chromosome of E. coli Y5. The ICEPmiChnXH1653 could be transferred between bacterial genera-within the order Enterobacteriales-from P. mirabilis to E. coli, indicating that the transmission of bla NDM-1 by ISCR1 elements or ICEs may be an important contributor to the carbapenem resistance development across species.
Bacterial conjugations. Conjugation experiments were carried out by filter mating with the rifampin-resistant E. coli EC600 as recipient. Overnight cultures of XH1653 and EC600 were mixed on an MH plate and incubated at 37°C for 18 h. The cells on the membrane were collected, resuspended in saline solution, and serially diluted before plating. Donors, recipients, and transconjugants were selected on MH agar plates containing 100 mg/liter rifampin and 100 mg/liter ampicillin. The successful transconjugants were identified by MALDI-TOF MS (bioMérieux, France), and the presence of bla NDM-1 , attL, int, and attR as the marker sequences of ICE in transconjugants was determined by PCR ( Table 3). The MIC profiles of the transconjugants were determined for differentiation between transconjugants and donor strains. The transconjugant was designated XH1814. The ICE transfer frequency was calculated as the number of transconjugants per donor cell.
Growth rate determination. Three independent cultures of EC600 and XH1814 were grown overnight and diluted to 1:100 in MH broth, and then aliquots were placed into a flat-bottom 100-well plate in three replicates. The plate was incubated at 37°C with agitation. The optical density at 600 nm (OD 600 ) of each culture was continuously determined for 20 h using a Bioscreen C MBR machine (Oy Growth Curves Ab Ltd., Finland). Growth rate was estimated based on OD 600 curves using an R script as previously described (46), and values returning a P value of ,0.05 from a Student t test were taken as significant.
Real-time quantitative PCR. The frequency of excision and mean copy number per cell, extrachromosomal copy number of the ICE, and the copy number of bla NDM-1 per chromosome were assessed by real-time quantitative PCR (37), using the formula as described previously (47). The genomic DNA was extracted using the QIAamp DNA minikit (Qiagen, USA), and quality and quantity of genomic DNA were  Table 3. Triplicate samples were included in each run, and qPCR experiments were performed in triplicate using TB Green Premix Ex Taq II (TaKaRa Bio) in a LightCycler 480 system (Roche, Switzerland). RNA-Seq. Three single colonies of EC600 and XH1814 were cultured overnight at 37°C in MH broth. Strains were diluted 1:100 in 100 ml of fresh MH broth and harvested at the mid-log growth phase. The cells were collected at 4°C using centrifugation (5,000 rpm, 10 min). Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) after liquid nitrogen grinding. Bacterial mRNA sequence library construction and sequencing were performed by Zhejiang Tianke (Hangzhou, China) (48). The sequenced reads were mapped to the EC600 genome and ICE sequence, respectively, using Rockhopper version 2.0.3 (49). The raw read count in output of Rockhopper was analyzed by the edgeR package (50). ggplot2 was used for figure generation (51).
Experimental evolution under nonselective conditions. Three single colonies of XH1814 and the EC600 ancestor strain were inoculated in MH broth without antibiotics and cultured under shaking (200 rpm) at 37°C. All evolved lineages were passaged daily. A 20-ml volume of overnight culture was collected and used for inoculation at a 1:100 dilution every day. Growth curves were performed every 5 days to assess the evolutionary changes.
Detection of carbapenem resistance loss. XH1814, three lineages of XH1814 at day 5, and three lineages of XH1814 at day 10 were incubated in MH broth at 37°C for 18 h. Overnight cultures were serially diluted before plating and were selected on an MH agar plate without antibiotics or with 0.25 mg/liter meropenem, respectively. The frequency of carbapenem resistance in XH1814 was calculated as the number of cells observed on an MH agar plate containing 0.25 mg/liter meropenem versus cells observed on an MH agar plate without antibiotics. The detection experiments were performed in triplicate.
Data availability. The complete genome sequences of P. mirabilis XH1653 and E. coli XH1815 isolates were deposited in GenBank under accession numbers CP065039 and CP069386, respectively. The RNA-Seq data from E. coli EC600 and E. coli XH1814 were deposited in GenBank under BioProject no. PRJNA699923.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. TABLE S1, PDF file, 0.1 MB.

ACKNOWLEDGMENTS
This work was supported by grants from the National Key Research and Development Program of China (2018YFE0102100) and the Zhejiang Provincial Medical and Health Science and Technology Plan (2018KY635).
There are no competing interests to declare.